Abstract
The CCCTC-binding factor CTCF is the only known vertebrate insulator protein and has been shown to regulate important developmental processes such as imprinting, X-chromosome inactivation and genomic architecture. In this study, we examined the role of CTCF in human embryonic stem cell (hESC) biology. We demonstrate that CTCF associates with several important pluripotency genes, including NANOG, SOX2, cMYC and LIN28 and is critical for hESC proliferation. CTCF depletion impacts expression of pluripotency genes and accelerates loss of pluripotency upon BMP4 induced differentiation, but does not result in spontaneous differentiation. We find that CTCF associates with the distal ends and internal sites of the co-regulated 160 kb NANOG-DPPA3-GDF3 locus. Each of these sites can function as a CTCF-dependent enhancer-blocking insulator in heterologous assays. In hESCs, CTCF exists in multisubunit protein complexes and can be poly(ADP)ribosylated. Known CTCF cofactors, such as Cohesin, differentially co-localize in the vicinity of specific CTCF binding sites within the NANOG locus. Importantly, the association of some cofactors and protein PARlation selectively changes upon differentiation although CTCF binding remains constant. Understanding how unique cofactors may impart specialized functions to CTCF at specific genomic locations will further illuminate its role in stem cell biology.
Highlights
Embryonic stem cells (ESCs) are derived from blastocysts and are considered pluripotent since they have the potential to give rise to a myriad of cell types
We find that CTCF associates with the distal ends of the coregulated 160 kb NANOG-DPPA3-GDF3 locus in human embryonic stem cell (hESC) and that each of these sites can serve as CTCF-dependent enhancerblocking insulator in a heterologous assay
We show that CTCF depletion significantly accelerates BMP4-induced differentiation and to a lesser extent TPA- and retinoic acid-induced differentiation pathways without inducing spontaneous differentiation
Summary
Embryonic stem cells (ESCs) are derived from blastocysts and are considered pluripotent since they have the potential to give rise to a myriad of cell types. For this reason they are of great therapeutic value. Embryonic stem cells have the ability to self-renew and proliferate indefinitely in culture and several studies have described the importance of the core regulatory circuitry that is comprised of NANOG, OCT4 and SOX2 proteins to maintain a pluripotent state [1,2]. Gene knockdown experiments in both mouse and human ESCs have shown that core transcriptional regulatory proteins such as subunits of the Mediator complex are important for activation of pluripotency genes such as OCT4 and NANOG [4,5]
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